Ubiquitin (Ub)-dependent proteolysis has been implicated in a variety of cellular processes, including cell cycle control, signal transduction, transcriptional regulation, DNA repair, receptor down-regulation, antigen presentation, and apoptosis. Abnormalities in the Ub system have been shown to cause pathological responses, including malignant transformation, and several genetic diseases. Ubiquitination of protein substrate involves a cascade of enzymatic reactions including the activation of Ub by Ub-activating enzyme, or El, the transfer to E2s (Ub-conjugating enzymes or Ubcs), and then to E3s (Ub protein ligases) for the tagging of Ub to the target. The E3s are the critical components that provide specificity to the Ub system by direct interaction with particular substrates. Itch, a novel E3 Ub ligase, is absent in the non-agouti-lethal 18H mice, or Itchy mice. These mice develop immunological and inflammatory diseases, including inflammation in the lung and stomach, hyperplasia in lymphoid and hematopoietic tissues, and constant itching in the skin, suggesting that Itch is involved in the regulation of immune responses. Itch contains an N-terminal protein kinase C-related C2 domain, four WW domains and C-terminal Hect ligase domain. At present, the biological pathways affected by Itch mutation have not been identified. Based on our preliminary studies, we propose that, first, Itch targets protein substrates for ubiquitination and sebsequent degradation and/or functional modification of the substrates, and Itch regulates signaling transduction pathways via the ubiquitination of its protein substrates; and, second, Itch deficient mice have a dysregulation of protein ubiquitination and of intracellular signal transduction. Our proposed plan is to test these hypotheses by the functional analysis of Itch-mediated ubiquitination of its substrates, by the examination of Itch-involved regulation of intracellular signal transduction in transiently transfected T cells and in Itch deficient primary T cells, and by the functional analysis of immunological and inflammatory responses in Itchy mice. The study on Itch is likely to shed light on the biological function of E3 ligases in the immune system, and may provide insights for therapeutic intervention in immunological diseases.